Emerging evidence implies that protein methyltransferases (PMTs) that play a crucial role in diverse biological processes and various human dieases including cancer. Protein N-terminal methyltransferase 1 (NTMT1/NRMT1) plays an important role in cell division, DNA repair, and chromatin interaction. NTMT1 is upregulated in various cancer patients' tissues, and its over- expression level ranks in the top 1% in both colorectal adenocarcinoma and malignant melanoma. Moreover, loss of NTMT1 results in cancer cell mitotic defects and sensitizes the DNA to chemotherapeutic agents. Therefore, NTMT1 is a compelling target for development of novel cancer therapeutics for colorectal cancer and melanoma. Therefore, it is of interest to develop novel small molecule inhibitors that might serve as biological probes as well as lead molecules for drug development. However, there are no specific small molecule probes available for NTMT1 to provide new insights into the functions of NTMT1 methyltransferase activity in tumorigenesis. To fill this gap, our long-term goal is to discover of novel, potent, and selective small molecule NTMT1 inhibitors. For this application, we will develop a series of facile and reproducible high-throughput screening (HTS) assays and run a pilot screen comprised of about 15,000 compounds to validate these assays in collaboration with the National Center for Advancing Translational Sciences Chemical Genomics Center (NCGC). In Aim 1, we will develop primary, counter, and secondary HTS-compatible biochemical NTMT1 assays in a quantitative HTS format. In Aim 2, we will perform pilot studies with selected libraries to identify small molecule inhibitors for NTMT1. In aim 3, we will characterize active compounds in structural, selectivity, and cell-based studies. Upon completion of this project we expect to define biochemical and cell based assays to screen large libraries of small molecules to identify potent and selective inhibitors of NTMT1. We anticipate that these studies will result in the identification of potent and selective first-in- class NTMT1 small molecule inhibitors for further development as chemical probes. The knowledge gained would expedite the development of NTMT1 modulators and our understanding of NTMT1-regulated pathways in cancer patients. The NCGC's top tier screening program and our experience in drug discovery integrate the expertise needed to be successful in such endeavor.